Abstract
Hepatoma cell lines are frequently used as in vitro alternatives to primary human hepatocytes. Cell lines are characterized by their unlimited life span, stable phenotype, high availability, and easy handling. However, their major limitation is the lower expression of some metabolic activities compared with hepatocytes. HepG2 is a human hepatoma that is most commonly used in drug metabolism and hepatotoxicity studies. HepG2 cells are nontumorigenic cells with high proliferation rates and an epithelial-like morphology that perform many differentiated hepatic functions. In this chapter, freezing, thawing, and subculturing procedures for HepG2 cells are described. We further provide protocols for evaluating lipid accumulation, glycogen storage, urea synthesis, and phase I and phase II drug metabolizing activities in HepG2 cells.
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References
Donato MT, Lahoz A, Castell JV et al (2008) Cell lines: a tool for in vitro drug metabolism studies. Curr Drug Metab 9:1–11
Donato MT, Jover R, Gomez-Lechon MJ (2013) Hepatic cell lines for drug hepatotoxicity testing: limitations and strategies to upgrade their metabolic competence by gene engineering. Curr Drug Metab 14:946–968
Aden DP, Fogel A, Plotkin S et al (1979) Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line. Nature 282:615–616
Knowles BB, Howe CC, Aden DP (1980) Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 209:497–499
Sormunen R, Eskelinen S, Lehto VP (1993) Bile canaliculus formation in cultured HEPG2 cells. Lab Invest 68:652–662
Fearn RA, Hirst BH (2006) Predicting oral drug absorption and hepatobiliary clearance: human intestinal and hepatic in vitro cell models. Environ Toxicol Pharmacol 21:168–178
Javitt NB (1990) Hep G2 cells as a resource for metabolic studies: lipoprotein, cholesterol, and bile acids. FASEB J 4:161–168
Dongiovanni P, Valenti L, Ludovica Fracanzani A et al (2008) Iron depletion by deferoxamine up-regulates glucose uptake and insulin signaling in hepatoma cells and in rat liver. Am J Pathol 172:738–747
Meier M, Klein HH, Kramer J et al (2007) Calpain inhibition impairs glycogen syntheses in HepG2 hepatoma cells without altering insulin signaling. J Endocrinol 193:45–51
Forte TM, McCall MR, Knowles BB et al (1989) Isolation and characterization of lipoproteins produced by human hepatoma-derived cell lines other than HepG2. J Lipid Res 30:817–829
Guo L, Dial S, Shi L et al (2011) Similarities and differences in the expression of drug-metabolizing enzymes between human hepatic cell lines and primary human hepatocytes. Drug Metab Dispos 39:528–538
Hilgendorf C, Ahlin G, Seithel A et al (2007) Expression of thirty-six drug transporter genes in human intestine, liver, kidney, and organotypic cell lines. Drug Metab Dispos 35:1333–1340
Jover R, Bort R, Gomez-Lechon MJ et al (1998) Re-expression of C/EBP alpha induces CYP2B6, CYP2C9 and CYP2D6 genes in HepG2 cells. FEBS Lett 431:227–230
Rodriguez-Antona C, Donato MT, Boobis A et al (2002) Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells. Xenobiotica 32:505–520
Wilkening S, Stahl F, Bader A (2003) Comparison of primary human hepatocytes and hepatoma cell line HepG2 with regard to their biotransformation properties. Drug Metab Dispos 31:1035–1042
Wilkening S, Bader A (2003) Influence of culture time on the expression of drug-metabolizing enzymes in primary human hepatocytes and hepatoma cell line HepG2. J Biochem Mol Toxicol 17:207–213
Lin J, Schyschka L, Mühl-Benninghaus R et al (2012) Comparative analysis of phase I and II enzyme activities in 5 hepatic cell lines identifies Huh-7 and HCC-T cells with the highest potential to study drug metabolism. Arch Toxicol 86:87–95
Bai J, Cederbaum AI (2004) Adenovirus mediated overexpression of CYP2E1 increases sensitivity of HepG2 cells to acetaminophen induced cytotoxicity. Mol Cell Biochem 262:165–176
Donato MT, Hallifax D, Picazo L et al (2010) Metabolite formation kinetics and intrinsic clearance of phenacetin, tolbutamide, alprazolam, and midazolam in adenoviral cytochrome P450-transfected HepG2 cells and comparison with hepatocytes and in vivo. Drug Metab Dispos 38:1449–1455
Tolosa L, Gomez-Lechon MJ, Perez-Cataldo G et al (2013) HepG2 cells simultaneously expressing five P450 enzymes for the screening of hepatotoxicity: identification of bioactivable drugs and the potential mechanism of toxicity involved. Arch Toxicol 87:1115–1127
Tang N, Wang Y, Wang X et al (2012) Stable overexpression of arginase I and ornithine transcarbamylase in HepG2 cells improves its ammonia detoxification. J Cell Biochem 113:518–527
McMillian MK, Grant ER, Zhong Z et al (2001) Nile red binding to HepG2 cells: an improved assay for in vitro studies of hepatosteatosis. In Vitro Mol Toxicol 14:177–190
Gomez-Lechon MJ, Donato MT, Martínez-Romero A et al (2007) A human hepatocellular in vitro model to investigate steatosis. Chem Biol Interact 165:106–116
Donato MT, Martínez-Romero A, Jimenez N et al (2009) Cytometric analysis for drug-induced steatosis in HepG2 cells. Chem Biol Interact 181:417–423
Donato MT, Gomez-Lechon MJ, Castell JV (1993) A microassay for measuring cytochrome P450IA1 and P450IIB1 activities in intact human and rat hepatocytes cultured on 96-well plates. Anal Biochem 213:29–33
Habig WH, Jakoby WB (1981) Assays for differentiation of glutathione S-transferases. Methods Enzymol 77:398–405
Malhi H, Barreyro FJ, Isomoto H et al (2007) Free fatty acids sensitise hepatocytes to TRAIL mediated cytotoxicity. Gut 56:1124–1131
Lowry OH, Rosebrough NJ, Farr AL et al (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Smith PK, Krohn RI, Hermanson GT et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85
Zawada RJ, Kwan P, Olszewski KL et al (2009) Quantitative determination of urea concentrations in cell culture medium. Biochem Cell Biol 87:541–544
Acknowledgements
The authors acknowledge the financial support of the Spanish Ministry of Health through grants PI10/0923 and PI13/0986 (Instituto de Salud Carlos III).
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Donato, M.T., Tolosa, L., Gómez-Lechón, M.J. (2015). Culture and Functional Characterization of Human Hepatoma HepG2 Cells. In: Vinken, M., Rogiers, V. (eds) Protocols in In Vitro Hepatocyte Research. Methods in Molecular Biology, vol 1250. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2074-7_5
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DOI: https://doi.org/10.1007/978-1-4939-2074-7_5
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